A subject of the present invention is new processes for the production of polyunsaturated fatty acid esters in a pure or concentrated form.
A more particular subject of the invention is processes for the production of esters of polyunsaturated fatty acids of the xcfx89-3 series starting with glycerides of fatty acids extracted from fish oils, phospholipids, or 1,2-dialkyene glycerols, using an enzymatic treatment.
A specific subject of the invention is a process for the production of glycerides of polyunsaturated fatty acids in a pure or concentrated form from fish oil or other sources, characterized in that it allows, by enzymatic treatment, a mixture to be obtained containing a high content of docosahexaenoic acid (DHA) and/or of eicosapentaenoic acid (EPA), which can reach, in the case of fish oils, 60%.
Also a subject of the invention is a process for the production of glycerides of polyunsaturated fatty acids, characterized in that phospholipids whose EPA and/or DHA content represents about 50% of the total fatty acids are obtained from natural phospholipids, in the presence of polyunsaturated fatty acids, by enzymatic treatment.
The invention also relates to a process for the production of synthetic glycerides, characterized in that a 1,2-dialkylene glycerol is subjected to an enzymatic action in the presence of concentrated or pure polyunsaturated fatty acids, in order to obtain a monoacylglyceride whose EPA and DHA content represents at least 70% of the total fatty acids.
The invention relates in particular to a process for obtaining polyunsaturated fatty acid concentrates which consists of subjecting a fish oil containing DHA and EPA to a selective enzymatic hydrolysis, in position 1,3 or 2, in order to obtain a mixture of free fatty acids, monoglycerides and diglycerides, separating the constituents of this mixture, collecting the free fatty acids which are purified by crystallization from urea, in order to increase the content of EPA and/or DHA, decomplexing the isolated fatty acids, carrying out an inter-esterification between the free fatty acids, concentrated into polyunsaturated fatty acids, and the crude oil, in the presence of a lipase specific for position or steric hindrance, in order to obtain a mixture enriched with polyunsaturated fatty acid glycerides, which is separated and freed from the free fatty acids.
In a preferred manner, sardine oil is used which is obtained by pressing fresh sardines caught in cold waters. The sardine offers the advantage of an easy and constant supply. On the contrary, tuna vagina bulbi oil, which is industrially exploited, although it has a higher EPA and DHA content, has the disadvantage that the supply of raw material is limited.
The effect of the initial enzymatic hydrolysis is to split the ester function of the glycerol esterified by a polyunsaturated acid and to leave intact the other ester functions according to Diagram A.
It is also possible to hydrolyze the triglycerides present in sardine oil by a non-specific lipase so as to obtain a mixture of free fatty acids in which the polyunsaturated fatty acids represent about 30% of the total mixture. This mixture of fatty acids is fractionated by physical means in order to give a mixture in which the polyunsaturated fatty acids and in particular EPA and DHA predominant, which can be up to 70-80% of the mixture of free fatty acids.
The free polyunsaturated fatty acids are reesterified in the presence of an enzyme and in particular in the presence either of a non-specific lipase or a lipase specific for position 2.
If a non-specific lipase is used, the glycerol will be reacted with the mixture of polyunsaturated free fatty acids and a triglyceride is obtained whose polyunsaturated fatty acid content is of the order of 60%.
If, using a lipase specific for position FA.2, a mixture of already-concentrated polyunsaturated fatty acids and a glyceride esterified in a single position by a polyunsaturated fatty acids in inter-esterified, a 1,2-diglyceride can be obtained in which only two positions are esterified by a polyunsaturated fatty acid.
It is also possible to hydrolyze a triglyceride, position 2 of which is esterified by a polyunsaturated fatty acid using a specific enzyme and in particular by a lipase of SN.2-specific type, in order to obtain a mixture of free polyunsaturated fatty acids, monoglycerides and diglycerides having a polyunsaturated fatty acid content comprised between 80 and 100%.
After fractionation of this mixture, the free fatty acids are purified by cold crystallization in the present of urea, in order to increase the EPA and DHA content. This concentrated mixture is then subjected to an inter-esterification by a triglyceride, position 2 of which is occupied by a polyunsaturated fatty acid, and positions 1,3 of which are occupied by a saturated fatty acid, in the presence of a 1,3-specific lipase, so as to obtain a triglyceride at least two hydroxyls of which are esterified by a polyunsaturated fatty acid.
The invention also relates to a synthesis process for triglycerides enriched with non-saturated fatty acids, which consists of saponifying fish oil by chemical route or by enzymatic route, in order to obtain a mixture of saturated and non-saturated fatty acids, converting the saturated fatty acids into lower alkyl esters in the presence of a selective lipase in order to obtain a mixture of alkyl esters of saturated fatty acids and of non-saturated fatty acids, separating the alkyl esters of the saturated fatty acid, collecting the free non-saturated fatty acids and reacting these fatty acids with glycerol in the presence of a specific lipase in order to obtain a mixture of triglycerides enriched with polyunsaturated fatty acids.
The attached diagrams illustrate these different variants of the process.
The enrichment of the crude sardine oil, by extraction of the polyunsaturated fatty acids (PUFA), from an average content of 30% to a fatty acid content corresponding to 50-60% of the total can be brought about according to one of the two schematized processes (A and B) according to the invention.
Until recently, the majority of the production of fish oil, considered to be a by-product of processing, was intended, after hydrogenation, for the production of magarine. The low added value of the product and the use of physico-chemical treatments for extraction, deodorization, decolouration and hydrogenation involving destruction of the properties of the oil, explains the lack of interest of manufacturers in this type of process.
The principle characteristic of fish oils is their high natural content (20-30%) of polyunsaturated fatty acids, of xcfx89 3 types, and in particular of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). At present, fish oil is the only commercially-exploitable source of long chain polyunsaturated fatty acids, despite production attempts using microbiological processes.
Since the epidemiological studies reported by Bang et al in 1971, and the physiological studies on this subject, it has become apparent that the essential fatty acids are the principle constituents of the phospholipids of the retina, of grey matter, of the epidermis; they play an important role in the central nervous system and have significant pharmacological properties in cardio-vascular diseases, as antithrombotics. Consequently, the interest shown by the pharmaceutical, cosmetic and parapharmaceutical industries has given rise to a fast-expanding market, in the USA, in JAPAN and in EUROPE. The PUFA (polyunsaturated fatty acids) market, based on the quality of the oils and therefore the added value, has 3 areas of use:
crude oils, up to 30% of PUFA, for uses in the agricultural and food industry (AFI), oleochemistry, biopolymers, animal fodder.
enriched oils, with 30 to 60%, for pharmaceutical and cosmetic uses.
purified PUFA""s, with 80 to 98%, for pharmacology.
At present, the grounds for objection of the fish oil industry reside at different levels, taking into account these new markets.
quality of the raw material
stability of the technical characteristics during the year, therefore necessitating a selection of the oils
extraction methods to be reconsidered
definition of the refining conditions as a function of requirements
storage conditions
Fish oils, sardine or cod liver, contain on average 20 to 30% of EPA+DHA relative to the total fatty acids. Among the wide variety of fish oils, the industrially-exploited tuna vagina bulbi oils are characterized by a higher content, up to 40%, but the supply of raw material is the main limitation to its production. Although it is easier to purify PUFA""s, free or in the form of ethyl esters, the requirements of the PUFA market, in particular pharmacology and cosmetics, relate exclusively to fatty acids in the natural form of triglycerides for reasons of efficiency. Starting with oils containing up to 30% of EPA and DHA, numerous techniques can be used on the crude oil, such as winterization, molecular distillation or crystallization using a solvent.
Due to the number of possible combinations of the position of the fatty acids on the triglycerides, it is necessary to use more complex techniques in order to obtain, from a crude oil, PUFA contents higher than 30%. However, the fractionation of the free or esterified fatty acids up to 65-80% is possible using a certain number of methods, such as extraction by supercritical fluid, complexing with urea, chromatography, separation on zeolite and even separation up to 90% is possible by HPLC.
Apart from these physico-chemical methods, enrichment by enzymatic route has been used with success on vegetable oils.
Physico-chemical techniques, due to their investment cost (for example 3MF for a 1 m3 industrial FSC extractor) can only be economically viable for enrichments greater than 80% characterized by a high added value. On the other hand, enzymatic techniques, which are more straightforward and therefore less costly, are in theory well suited to fill the gap between the crude oil and on oil titrating 55-60% PUFA.
In fact, for some years, numerous studies have shown the physiological and dietetic importance of polyunsaturated fatty acids and especially of eicosapentaenoic acid (or EPA=xcex94-5, 8, 11, 14, 17-eicosapaentaenoic acid) and docosahexaenoic acid (or DHA=xcex94-4, 7, 10, 13, 16, 19-docosahexaenoic acid). These compounds are only formed in the human body in very small quantities and the quantities produced according to the two biological diagrams hereafter carry on decreasing over the years, such that a deficiency may result in pregnant women and in elderly people. The diagram hereafter summarizes the formation pattern of polyunsaturated fatty acid in the body.
There are many uses of enzymes in the treatment of fish, production of protein hydrolyzates by proteases, silage making, breaking down of tuna, tenderizing of the flesh, hydrolysis of the tissues surrounding the eggs (caviar), elimination of the liver membranes. With regard to the oils, numerous publications show the usefulness of enzymatic treatments for extracting and purifying the polyunsaturated fatty acids. The most commonly used enzymes are lipases which either allow the triglycerides to be hydrolyzed into fatty acids and glycerol under aqueous conditions, or conversely the fatty acids to be esterified under anhydrous conditions or in the presence of organic solvents.
Numerous lipases hydrolyze the triglycerides according to the following sequence: 
However, certain lipases do not respond to these kinetics due to the specific xe2x80x9crecognitionxe2x80x9d:
of certain fatty acids
of the position of these fatty acids on the triglyceride
of glycerides of different molecular weights or
of their stereospecificity.
Thus numerous lipases of different specificities exist:
intestinal lipases or lipases of panniculus adiposus, of insect muscle, which preferably hydrolyze the monoglycerides rather than the triglycerides.
lipases of Mucor, Rhizopus, mil, and porcine pancreatic lipase, which are specific for positions 1,3 on the triglyceride, whilst the lipase of Pseudomonas is specific for position 2.
lipase of Candida which preferably hydrolyzes the ester bonds of DHA.
lipase of cabbage (B. napus) which recognizes the position of the double bond on the fatty acids and differentiates -linolenci acid from DHA, or lipase of Geotrichum which is specific for fatty acids containing CIS-9 and CIS-9 -CIS-12 unsaturations.
This specificity of the lipases is generally the basis of the fractionation processes for commercially useful fatty acids contained in vegetable or fish oils.
Thus the following enrichment processes have been described: of xcex1-linolenic acid in a primrose oil (from 9.5% to 64.6%) with a cabbage lipase; of DHA in a cod liver oil (from 12.7 to 45.9%) with a Mucor lipase; of EPA+DHA in a cod liver oil (from 23 to 72.2%) with a Mucor lipase; of DHA in a tuna oil (concentration factor=3) with a Candida lipase; of 5-eicosaenoic acid (20:1) in a Meadowfoam oil (from 83.5 to 95%) by a Chromabacterium lipase.
Schematically, the preparation of triglycerides enriches with PUFA is based on an inter-esterification catalyzed by a selective lipase: 
However, the prior enzymatic processes require a supply of free or esterified PUFA of high purity. Consequently, this supply must be preceded by a total hydrolysis of the triglycerides in order to release the free PUFA""s by a lipase and by a purification by chemical route or using specific lipases.
There are particular difficulties which the present process aims to resolve.